Why Proximity to Cysteine Does Not Necessarily Lead to Covalent Bond Formation: Warhead Reactivity, Reaction Geometry, and Target-Site Microenvironment

Introduction

Cysteine is one of the amino acid residues most frequently exploited in targeted covalent drug discovery. Its side-chain thiol has substantial nucleophilic potential and can form stable covalent bonds with electrophilic groups such as acrylamides. Targeted covalent inhibitors typically first rely on the ligand scaffold to form a specific noncovalent complex with the protein and then use an appropriately positioned covalent reactive group (CRG), commonly referred to as a covalent warhead, to capture a cysteine located near the binding site.[1]

Observing a warhead in close proximity to a cysteine in a crystal structure does not directly imply that the two will necessarily form a covalent bond. Covalent bond formation is jointly controlled by three key factors: the intrinsic reactivity of the covalent warhead, the reaction geometry established after ligand binding, and the reactivity of the target cysteine within the local protein microenvironment.

The study of c-KIT covalent inhibitors by McAulay et al. provides an experimental example. Acrylamide compound 25, designed to target Cys788, remained noncovalently bound to c-KIT; after the warhead was replaced with an alkynyl benzoxazine, compound 26 formed a covalent bond with the same Cys788 in the crystal structure. When the same compound 26 bound to the structurally related KDR kinase, the warhead underwent a pronounced conformational flip and remained noncovalently bound.[4] This set of structural comparisons connects warhead reactivity, spatial orientation, and the protein microenvironment within the same experimental system.

Key Abbreviations and Structural Identifiers Used in This Article

Abbreviation or Identifier	Full Name or Meaning	Specific Meaning in This Article
Cys / Cys788	Cys is the three-letter abbreviation for cysteine; the number indicates the residue position in the protein sequence	Cys788 refers to cysteine residue 788 in c-KIT and is the target residue discussed in this article for the covalent bond-forming behavior of compounds 25 and 26.
c-KIT	KIT receptor tyrosine kinase, also known as CD117	The principal example protein in this article. Compounds 25 and 26 both bind to its kinase domain and are used to compare the effects of different warheads and binding conformations on covalent capture of Cys788.
KDR / VEGFR2	KDR: kinase insert domain receptor; VEGFR2: vascular endothelial growth factor receptor 2	The protein encoded by KDR is VEGFR2. It is compared with c-KIT to illustrate how the same compound 26 can adopt different warhead conformations and produce different bond-forming outcomes in different protein-binding environments.
DFG-in / DFG-out	DFG refers to the conserved Asp–Phe–Gly motif in protein kinases; “in” and “out” denote different conformational states of this motif	The c-KIT Cys788 reactivity calculations cited in this article involve both DFG-in and DFG-out conformations. The DFG-out conformation is also commonly observed in type II kinase inhibitor-bound states.
PDB	Protein Data Bank	A database for storing and retrieving experimentally determined three-dimensional structures of biological macromolecules. 6XVA, 6XVB, and 6XVK discussed in this article are all PDB structure identifiers.
6XVA / 6XVB / 6XVK	Three PDB identifiers corresponding to the key structural evidence discussed in this article	6XVA: c-KIT with compound 25, in which the target Cys788 does not form a covalent bond with the ligand; 6XVB: c-KIT with compound 26, in which compound 26 forms a covalent bond with Cys788; 6XVK: KDR (VEGFR2) with compound 26, in which compound 26 remains noncovalently bound and adopts a different warhead orientation.

1. Covalent Inhibition Involves Two Stages: Molecular Recognition and Chemical Reaction

A typical two-step irreversible covalent inhibition process can be simplified as:

P + L ⇌ P·L → P–L

Here, P represents the protein, L the ligand, P·L the pre-reaction noncovalent protein–ligand complex, and P–L the covalent adduct formed after covalent bond formation.

The first stage is controlled by conventional molecular recognition. After entering the binding pocket, the ligand forms a noncovalent complex through shape complementarity, hydrogen bonding, electrostatic interactions, hydrophobic interactions, and other forces.

The second stage is the chemical reaction. The covalent warhead, already positioned within the binding site, reacts with the target residue and converts the noncovalent complex into a covalent adduct.

For irreversible inhibitors that conform to this two-step model, K_I describes an apparent binding parameter associated with formation of the pre-reaction complex, k_inact represents the maximum first-order rate constant for conversion of this complex into the covalently inactivated state, and k_inact/K_I is commonly used to evaluate irreversible inhibition efficiency.[5]

These two stages can be partially separated. Even if two compounds have similar noncovalent binding affinities, differences in the rate of covalent bond formation can lead to substantial differences in time-dependent inhibition and target occupancy. Conventional endpoint IC₅₀ values are also influenced by incubation time, substrate concentration, and experimental conditions. Therefore, for irreversible covalent inhibitors, a single IC₅₀ value generally cannot fully describe both noncovalent binding and the subsequent covalent reaction.[5]

2. Cysteines in Proteins Exhibit Different Reactivities

Cysteine itself has substantial nucleophilic potential, but cysteine residues located in different proteins or at different positions do not constitute a chemically uniform set of reactive sites.

Using quantitative reactivity-based proteomics, Weerapana et al. found that cysteine residues within proteins vary considerably in their reactivity toward electrophilic probes. Highly reactive cysteines represent only a fraction of detected sites and are often found at catalytic, redox-regulatory, and other functional sites.[2]

For typical cysteine nucleophilic reactions, the deprotonated thiolate is generally more nucleophilic than the neutral thiol. The acid–base properties of the cysteine side chain can therefore affect reaction rates, but pKₐ is not the sole determinant. The local electrostatic environment, hydrogen-bonding network, solvent exposure, side-chain conformation, and orientation of the nucleophile relative to the warhead can all influence the final reaction efficiency.[3]

Computational analyses by Awoonor-Williams and Rowley of potentially covalently targetable cysteines in protein kinases likewise showed substantial differences among sites in predicted acid–base properties and local reaction environments.[3]

Therefore, the presence of a Cys at a spatially suitable position within a binding pocket identifies only a potential covalent target. Further design must consider the chemical state of that cysteine in the specific protein conformation and whether it can adopt a geometry suitable for nucleophilic attack.

3. Covalent Warheads Require an Appropriate Reactivity Window

Increasing the electrophilicity of a warhead generally enhances its tendency to react with sulfur nucleophiles, but targeted covalent drugs require this reactivity to be controlled.

If reactivity is too low, the warhead may fail to achieve effective covalent capture within the available binding time even when the ligand has positioned it close to the target cysteine. If reactivity is too high, the compound may react excessively rapidly with glutathione (GSH), cysteines in other proteins, and other biological nucleophiles, thereby increasing nonspecific adduct formation and off-target risk.

During the development of new cysteine-targeting warheads, McAulay et al. systematically compared the reactions of different structures with GSH as well as their stability in pH 7.4 buffer. Some early electrophilic heterocycles reacted rapidly with GSH while also exhibiting poor stability. The subsequently developed alkynyl benzoxazine 5e showed a more balanced profile in the same experimental system, with a GSH reaction half-life of 82 min and a half-life of more than 10,000 min in buffer.[4]

This result illustrates the concept of a reactivity window: a covalent warhead must retain sufficient chemical reactivity for the favorable environment provided by the target protein to promote bond formation while maintaining adequate chemical stability to reduce nonselective reactions in complex biological systems.

The GSH reaction half-life is useful for comparing the relative reactivities of a series of warheads under standardized experimental conditions, but it cannot serve as a universally applicable “optimal value” across different assay systems. The result is influenced by compound concentration, GSH concentration, pH, temperature, analytical method, and other factors.[4]

4. c-KIT Cys788: Why the Acrylamide Failed to Form the Expected Covalent Bond

McAulay et al. began with a kinase inhibitor scaffold capable of binding c-KIT. Existing structural data showed that one side chain of this scaffold pointed toward Cys788, and the researchers therefore used this position to introduce a cysteine-reactive group.[4]

The resulting compound 25 contained a classical acrylamide Michael acceptor. The design objective was to position the acrylamide close to Cys788, thereby enabling nucleophilic addition of the thiol to the α,β-unsaturated amide.

X-ray crystallography showed that compound 25 remained noncovalently bound to c-KIT. The corresponding structure is PDB 6XVA. The acrylamide side chain has considerable conformational freedom and, in the actual binding site, bends backward away from Cys788 and interacts with the local water network.[4]

This result demonstrates the need to distinguish between two structural factors: spatial proximity and reaction geometry:

Spatial proximity describes whether the warhead can reach the vicinity of the target residue;

Reaction geometry describes whether the nucleophilic atom and electrophilic center can be arranged at a direction and distance suitable for bond formation.

For reactions such as Michael addition, which have defined orbital and geometric requirements, merely positioning the warhead near the cysteine is insufficient. Linker flexibility, the water network, and local noncovalent interactions can all stabilize a binding conformation that is unfavorable for chemical reaction.

The structure of compound 25 therefore directly demonstrates that a covalent warhead must maintain an effective reactive orientation in the bound state, rather than being evaluated solely on the basis of static distances in a design model.

5. Alkynyl Benzoxazine Simultaneously Changes Reactivity and Reactive Conformation

The researchers subsequently replaced the acrylamide with a conformationally constrained alkynyl benzoxazine warhead to obtain compound 26.

This design was initially based on two considerations. First, the new warhead had a thiol reactivity distinct from that of a conventional acrylamide. Second, the cyclic structure increased local rigidity, helping to maintain a reactive vector directed toward the target residue. The authors explicitly proposed “fixing the reactive direction of the Michael acceptor through a rigid structure” as one of the design hypotheses.[4]

X-ray crystallography ultimately showed that compound 26 formed a covalent bond with c-KIT Cys788, corresponding to PDB 6XVB.[4] The RCSB PDB description of this structure likewise explicitly identifies covalent binding to Cys788.

In the same experimental system, compound 26 had a GSH reaction half-life of approximately 65 min, and its GSH reactivity was approximately twice that of the corresponding acrylamide 25.[4] The authors also cited calculations by Awoonor-Williams and Rowley for c-KIT Cys788: in the DFG-in and DFG-out conformations examined, the thiol pKₐ of this site was predicted to be greater than 21, suggesting a high deprotonation barrier to formation of the more strongly nucleophilic thiolate.[3,4]

The pKₐ > 21 value here is a prediction obtained from a specific computational model rather than an experimentally measured value for Cys788. It was used to explain why the authors considered increasing warhead reactivity and should not be interpreted as a direct experimental determination of the actual pKₐ of Cys788 in the protein environment.

From compound 25 to compound 26, at least two factors changed simultaneously:

① The intrinsic reactivity of the warhead toward sulfur nucleophiles increased;

② The conformational constraint and reactive orientation of the warhead in the protein-bound state changed.

Therefore, the successful covalent capture achieved by compound 26 cannot be attributed solely to “increased electrophilicity.” The explanation proposed in the original study involves the combined effects of the low nucleophilicity of the target cysteine, warhead reactivity, and a rigid reactive vector.[4]

The enzyme activity data in the study also provide an important boundary for interpreting the evidence. At 300 μM ATP, the c-KIT IC₅₀ of compound 25 was 0.240 μM, whereas that of compound 26 was 2.08 μM; although compound 26 formed a covalent bond in the co-crystal structure, its enzymatic IC₅₀ was not lower under these conditions.[4]

The authors also noted that the protein and compound concentrations used for co-crystallization were high, which may have promoted covalent bond formation under crystallization conditions, whereas the extent of bond formation may not have been identical under the conditions used for the in vitro activity assay.[4] Thus, 6XVB provides clear structural evidence of covalent bond formation, but this structure alone cannot establish that compound 26 achieves the same degree of irreversible target occupancy under all biochemical or cellular conditions.

6. The Same Compound Adopts Different Reactive Conformations in c-KIT and KDR

Structural comparison of compound 26 bound to c-KIT and KDR further demonstrates that the local protein environment can directly alter the spatial orientation of the warhead.

The original study first compared the conformations of corresponding cysteines in the two kinases, namely c-KIT Cys788 and KDR Cys1024. Analysis of existing PDB structures showed that Cys788 pointed toward the active site in most of the analyzed c-KIT structures, whereas Cys1024 pointed away from the pocket in approximately 80% of the analyzed KDR structures. The neighboring residues and pocket shapes of the two binding sites also differed.[4]

In the structure of compound 26 bound to c-KIT, the warhead forms a covalent bond with Cys788; in the KDR structure PDB 6XVK, compound 26 remains noncovalently bound.[4]

The quinoline and pyridine cores maintain similar binding interactions in the two complexes, but the benzoxazine warhead is rotated by approximately 180° in KDR relative to its orientation in the c-KIT structure. This flip positions the terminal methylalkyne group within a hydrophobic pocket of KDR, moving it away from the reactive orientation that enables covalent capture in c-KIT.[4]

Thus, covalent selectivity can also arise from the ability of the protein-binding pocket to select among different warhead conformations:

The protein-binding pocket not only determines whether a ligand can bind, but can also control whether the subsequent chemical reaction has a suitable geometry by stabilizing different warhead conformations.

Even when two related proteins contain potentially targetable cysteines at corresponding positions, the same covalent warhead may produce different bond-forming outcomes. Local pocket geometry, neighboring residues, the water network, and ligand conformation collectively determine the actual reactive conformation and covalent bond-forming outcome.

7. Covalent Activity Requires an Evidence Chain from Chemical Reactivity to Cellular Selectivity

Evaluation of targeted covalent compounds requires distinction among three levels: intrinsic warhead reactivity, covalent protein modification, and selectivity in biological systems.

7.1 Warhead Reactivity and Stability

Reactions with model thiols such as GSH can be used to compare the tendency of warheads to react with biologically relevant nucleophiles; buffer stability is used to determine whether a compound is prone to hydrolysis or other degradation in the absence of the target nucleophile.

These experiments address how readily the warhead itself undergoes chemical reaction and cannot independently establish target selectivity.

7.2 Covalent Adduct Formation with Intact Protein

Intact-protein mass spectrometry can detect whether treatment with a compound produces a mass increase corresponding to the ligand and is an important method for confirming an irreversible covalent hit.[1]

This experiment can demonstrate formation of a stable protein–ligand adduct, but the mass increase alone generally cannot identify the specific modified residue.

7.3 Covalent Modification Site

Proteolytic digestion combined with peptide mass spectrometry and MS/MS can further localize the modified amino acid residue and thereby determine whether the covalent reaction occurred at the expected Cys site.[1]

7.4 Structural Validation

X-ray crystallography can simultaneously provide the ligand-binding mode, warhead orientation, and spatial environment surrounding the target residue. Structural comparisons of compounds 25 and 26 in c-KIT and compound 26 in KDR demonstrate the direct value of structural biology for distinguishing between “being close to the target residue” and “forming an effective reactive conformation.”[4]

7.5 Cellular and Proteome-Wide Selectivity

After entering cells, a covalent warhead encounters numerous potential nucleophilic sites. Lanning et al. used activity-based proteomic profiling combined with quantitative mass spectrometry to systematically investigate the proteome-wide selectivity of covalent kinase inhibitors and proposed that different compounds exhibit corresponding selectivity windows; once these windows are exceeded, broad proteome reactivity and cell death unrelated to the primary kinase target can occur.[6]

The goal of targeted covalent design is therefore to ensure that effective covalent reaction at the target site markedly outcompetes reactions with unrelated proteins under relevant exposure conditions.

8. Design Sequence from Reactive Site to Covalent Compound

8.1 First Evaluate the Target Cysteine

The location, solvent exposure, local conformation, and microenvironment of a cysteine determine whether it provides exploitable reaction conditions. The presence of a Cys in the protein sequence, or a short distance between a Cys and a ligand in a crystal structure, is insufficient on its own to establish covalent targetability.[2–4]

8.2 Then Select Warhead Reactivity

When the target residue has low reactivity, a warhead that is too mild may fail to achieve a sufficient bond-forming rate; excessively high warhead reactivity, however, increases nonspecific reaction. GSH reactivity and chemical stability can be used to establish a relative reactivity range within the same series of compounds.[4]

8.3 Simultaneously Optimize the Reactive Vector

The attachment position, linker length, conformational flexibility, and ring-system rigidity determine the position and orientation of the warhead in the bound state. The structural differences between c-KIT compounds 25 and 26 demonstrate that the reactive vector can directly determine whether an electrophilic group that has already entered the correct pocket can complete covalent bond formation.[4]

8.4 Finally Validate Genuine Covalent Selectivity

Intact-protein mass spectrometry, peptide mass spectrometry, structural biology, and cellular and proteome-level evaluations together constitute the evidence chain for validating covalent selectivity; the individual validation levels are described in Section 7.[1,6]

For compounds that conform to the two-step irreversible inhibition model, K_I, k_inact, and k_inact/K_I further distinguish pre-reaction binding from subsequent covalent inactivation.[5]

9. Representative Chemicals Related to Cysteine-Targeted Covalent Bond Formation, Warhead Reactivity, and Validation Studies

Table 1. Reagents for Evaluating Cysteine Reactivity, Thiol State, and Reactivity

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
Target cysteine nucleophile model	52-90-4	C108238	L-Cysteine	Animal-origin free, Moligand™, for cell culture, ≥98%	Contains a free thiol and can be used to study cysteine nucleophilic reactions, thiol ionization, and reaction rates with electrophilic warheads, as well as to compare cysteine covalent-capture capability under different conditions.
Physiological thiol reactivity model	70-18-8	G774620	Reduced glutathione	PharmPure™, European Pharmacopoeia (Ph.Eur)	An important intracellular low-molecular-weight thiol that can be used to evaluate covalent warhead thiol reactivity, reaction half-life, and nonspecific reaction tendency, and is a commonly used model for establishing a warhead reactivity window.
Low-molecular-weight thiol competition model	616-91-1	A105421	N-Acetyl-L-cysteine (NAC)	PharmPure™, USP, Moligand™, European Pharmacopoeia (Ph.Eur), ≥98.5%	Retains a reactive thiol and can be used for thiol trapping, competition reactions, and validation of covalent interactions involving electrophilic compounds, as well as for comparing the reactivity of different electrophilic structures toward low-molecular-weight sulfur nucleophiles.
Thiol-containing reducing agent	3483-12-3	D104860	DL-Dithiothreitol (DTT)	Molecular biology grade, ≥99%	Used to reduce protein disulfide bonds and maintain cysteine residues in the reduced state; however, DTT itself contains free thiols and can compete with electrophilic warheads. Its concentration should therefore be controlled or it should be removed before covalent-binding experiments. It can also serve as a control for competitive reactions with electrophilic warheads.
Non-thiol reducing agent	51805-45-9	T755741	Tris(2-carboxyethyl)phosphine hydrochloride (TCEP HCl)	UltraBio™, ≥98% (NMR)	A phosphine-based disulfide-reducing reagent that can be used to maintain protein cysteines in the reduced state while avoiding the introduction of additional free thiols as competing nucleophiles, making it suitable for covalent-binding and cysteine-reactivity studies.
Free thiol quantification reagent	69-78-3	D105559	5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB)	≥98%	A classical reagent for quantifying free thiols that can be used to measure reactive thiol content in proteins or small molecules and to compare changes in cysteine thiols before and after covalent modification.

Table 2. Reagents for Cysteine-Targeted Covalent Warheads and Electrophilic Reaction Studies

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
Acrylamide-type electrophilic group model	79-06-1	A108469	Acrylamide	Moligand™, ultrapure grade, ≥99.9%	A basic α,β-unsaturated amide structure that can be used to study thiol conjugate addition to electrophilic alkenes and the reactivity basis of acrylamide-type covalent warheads.
Acrylamide warhead synthesis reagent	814-68-6	A104614	Acryloyl chloride	≥96%, contains 200 ppm MEHQ stabilizer	Can be used for acryloylation of amino-containing molecular scaffolds to introduce α,β-unsaturated amide electrophilic groups and is suitable for synthetic studies of covalent ligands and cysteine-targeting molecules.
Chloroacetamide-type electrophilic reagent	79-07-2	C106072	Chloroacetamide	≥99%	A haloacetamide electrophilic structure that can react with sulfur nucleophiles through nucleophilic substitution and is suitable for studies of cysteine-targeted covalent fragments, warhead reactivity, and leaving-group effects.
Bromoacetamide-type electrophilic reagent	683-57-8	B139173	2-Bromoacetamide	≥98% (GC)	A bromoacetamide electrophilic model that can be used to compare the effects of halogen leaving-group variation on thiol alkylation rates and covalent reactivity.
Iodoacetamide-type cysteine-labeling reagent	144-48-9	I131590	Iodoacetamide	Moligand™, ultrapure grade, ≥99% (NMR)	Can alkylate free cysteine thiols and is commonly used for blocking protein cysteines, site labeling, and proteomics sample preparation. It can serve as a reference high-reactivity cysteine electrophile.
Maleimide-type thiol-reactive reagent	128-53-0	E100553	N-Ethylmaleimide (NEM)	Moligand™, ultrapure grade, ≥99%	Its electron-deficient double bond can undergo conjugate addition with thiols. It is commonly used to block free protein thiols and can be used to compare electrophile reaction rates and cysteine accessibility.
Maleimide-type electrophilic scaffold	541-59-3	M100788	Maleimide	≥98%	Contains an activated double bond and can serve as a basic electrophilic scaffold for thiol conjugate addition and cysteine-selective modification, enabling studies of the relationship between warhead electronic properties and thiol reactivity.

Table 3. Cysteine-Targeted Covalent Inhibitors and Compounds for Target-Selectivity Studies

Category	CAS No.	Aladdin Cat. No.	Name	Specification or Purity	Product Features and Applications
EGFR Cys797 covalent inhibitor	1421373-65-0	A302849	Osimertinib (AZD9291)	Moligand™, ≥99%	Covalently captures EGFR Cys797 through an electrophilic group and can be used to study spatial positioning of acrylamide-type warheads, time-dependent inhibition, and target selectivity.
BTK Cys481 covalent inhibitor	1420477-60-6	A413911	Acalabrutinib (ACP-196)	Moligand™, ≥98%	Covalently targets Bruton's tyrosine kinase Cys481 through an alkynamide-type electrophilic group and can be used to compare the reactivity, binding orientation, and covalent target occupancy of non-acrylamide warheads.
KRAS G12C covalent inhibitor	2326521-71-3	A414244	Adagrasib (MRTX849)	Moligand™, ≥98%	Covalently captures Cys12 in mutant KRAS G12C and can be used to study mutant-site selectivity, pocket preorganization, electrophilic warhead positioning, and irreversible target occupancy.
BTK Cys481 acrylamide-type inhibitor	936563-96-1	P127143	Ibrutinib (PCI-32765)	Moligand™, ≥98%	Contains an acrylamide-type electrophilic group that covalently modifies Bruton's tyrosine kinase Cys481 and is suitable for studying the two-step process of reversible recognition followed by covalent bond formation.
KRAS G12C covalent-binding research compound	2252403-56-6	S414293	Sotorasib (AMG510) racemate	Moligand™, ≥97%	Corresponds to a KRAS G12C covalent inhibitor scaffold and can be used for studies of Cys12 covalent capture, warhead–pocket matching, and structure–reactivity relationships; the racemic composition can be used to evaluate the influence of stereochemistry on molecular recognition and reaction behavior.
EGFR-family covalent inhibitor	850140-72-6	A409277	Afatinib (BIBW2992)	10 mM in DMSO	Contains an activated unsaturated amide-type electrophilic group capable of irreversibly modifying corresponding cysteine residues in the EGFR family and is suitable for studies of covalent-binding kinetics, target occupancy, and reactivity–selectivity relationships.

Note: The products listed above are representative Aladdin products related to scientific research. Specific uses should be determined according to the product specification, batch COA, and the intended reaction or evaluation system. Additional product specifications, grades, and COA information can be searched on the Aladdin website using the product name, CAS number, or catalog number.

References

[1] Lucas, S. C. C.; Blackwell, J. H.; Hewitt, S. H.; Semple, H.; Whitehurst, B. C.; Xu, H. Covalent hits and where to find them. SLAS Discovery 2024, 29(3), 100142. DOI: 10.1016/j.slasd.2024.01.003.

[2] Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.; Dillon, M. B. D.; Bachovchin, D. A.; Mowen, K.; Baker, D.; Cravatt, B. F. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 2010, 468, 790–795. DOI: 10.1038/nature09472.

[3] Awoonor-Williams, E.; Rowley, C. N. How Reactive are Druggable Cysteines in Protein Kinases? Journal of Chemical Information and Modeling 2018, 58(9), 1935–1946. DOI: 10.1021/acs.jcim.8b00454.

[4] McAulay, K.; Hoyt, E. A.; Thomas, M.; Schimpl, M.; Bodnarchuk, M. S.; Lewis, H. J.; Barratt, D.; Bhavsar, D.; Robinson, D. M.; Deery, M. J.; Ogg, D. J.; Bernardes, G. J. L.; Ward, R. A.; Waring, M. J.; Kettle, J. G. Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors. Journal of the American Chemical Society 2020, 142(23), 10358–10372. DOI: 10.1021/jacs.9b13391.

[5] Miyahisa, I.; Sameshima, T.; Hixon, M. S. Rapid Determination of the Specificity Constant of Irreversible Inhibitors (k_inact/K_I) by Means of an Endpoint Competition Assay. Angewandte Chemie International Edition 2015, 54(47), 14099–14102. DOI: 10.1002/anie.201505800.

[6] Lanning, B. R.; Whitby, L. R.; Dix, M. M.; Douhan, J.; Gilbert, A. M.; Hett, E. C.; Johnson, T. O.; Joslyn, C.; Kath, J. C.; Niessen, S.; Roberts, L. R.; Schnute, M. E.; Wang, C.; Hulce, J. J.; Wei, B.; Whiteley, L. O.; Hayward, M. M.; Cravatt, B. F. A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors. Nature Chemical Biology 2014, 10, 760–767. DOI: 10.1038/nchembio.1582.

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